Method for reducing the content of pathogenic organisms present in food materials

ABSTRACT

The present invention relates to a method for reducing the concentration of pathogenic organisms such as  Listeria  spp. in fermented food products. The method comprises the steps of: (i) providing a food material, (ii) mixing said food material with a starter culture, (iii) mixing the food material with at least one adjunct culture in form of a bacteriocin-producing  Pediococcus  species, (iv) subjecting the mixture provided in step (iii) to a fermentation process, said fermentation process being conducted at conditions that are sub-optimal for growth of the bacteriocin-producing  Pediococcus  species in order to provide a limited acidification and allow for a high production of bacteriocin, and obtain a fermented food product. Furthermore, the invention relates to the use of bacteriocin-producing  Pediococcus  species as an adjunct culture for securing microbial safety of fermented food products.

FIELD OF INVENTION

The present invention relates to the field of improving microbial safetyin the production of food products. In particular the present inventionrelates to microbial strains useful for reducing the amount ofpathogenic organisms e.g., Listeria when added to a fermented foodproducts, such as a fermented meat product

PRIOR ART

During the production of fermented food products, such as e.g. sausageproducts, a starter culture is most often applied in order to controlthe fermentation process instead of relying on the natural developingflora. Commonly, the starter culture comprises a combination of one ormore lactic acid bacteria (LAB) and one or more species from theMicrococcaceae and Staphylococcaceae families. During the fermentationprocess the lactic acid bacteria primarily produce lactic acid wherebypH drops to the desired pH-value depending on the culture and theprocessing conditions (temperature, sugar type/content etc.) and thefood product produced.

Whereas the lactic acid bacteria are mainly responsible for the acidformation, the Micrococcaceae spp. and Staphylococcaceae spp. areresponsible for enhancing the flavour formation by producingnon-volatile and volatile compounds through various biochemical reactionsteps. Additionally, the Micrococcaceae spp. and Streptococcaceae spp.are responsible for the speed and intensity of colour formation inparticular in fermented sausage types.

Micrococcaceae spp. and Streptococcoceae spp. are very sensitive to lowpH as their growth is drastically slowed down when pH is reduced to a pHbelow 5.0. In e.g. the manufacturing of dried sausages it is essentialfor the flavour and colour formation of the meat products that theacidification profile is well-controlled and is not altered from batchto batch. In particular, a fast pH-lowering may impair the quality andresult in a less mature and less complex flavour profile that will forcethe food product manufacturer to ripen the food product for a longerperiod of time to reach the same flavour intensity (Tjener et al.,2003).

In manufacturing of fermented food products presence of pathogenicorganisms like Listeria monocytogenes may be a problem if the rawmaterials are contaminated. During the production of e.g. fermentedsausages Listeria monocytogenes will normally decrease in numbers duringthe fermentation and ripening period, primarily, due to the formation oflactic acid, the resulting drop in pH and due to the reduction in wateractivity caused by the subsequent drying process. However, quite often,a considerable number of Listeria monocytogenes survives. This may causea serious safety problem as consumption of infected food may give riseto lethal listerial infections (listeriosis).

In order to reduce the presence of pathogenic micro-organisms in thefood product certain bacteriocin producing lactic acid bacteriaincluding Pediococcus strains and certain Lactobacillus strains havebeen added to the starter culture to produce bacteriocins some of whichkill and/or inactivate the pathogenic organisms and accordingly reducetheir concentration in the product.

Foegeding et al. (1992) disclose the effectiveness of pediocin producedin situ by Pediococcus acidilactici as an antilisterial component.However, the fermentation of sausages was conducted at 38° C. whichcaused an extensive acid production and thus a very fast drop in pH. Asmentioned above a rapid pH-lowering impairs the general quality of theproduct and results in a less mature and less complex flavoured profile.Thus, the adverse influence of Pediococcus acidilactici, on the generalquality of the resulting product renders the method unsuitable for foodfermentations where the above-mentioned conditions and features apply.

Utility model BA 1994 00266 discloses a lactic acid bacterial starterculture comprising a selected bacteriocin-producing Pediococcus spp. anda selected bavaricin-producing Lactococcus useful for inhibitingpathogenic organisms e.g. Listeria in meat products including fermentedmeat products.

Evidently, Pediococcus spp. are often not well qualified as startercultures in the manufacturing of food products although the species areknown for their potential for reducing the amount of pathogenicorganisms e.g. Listeria.

Thus, there is a persisting need in the industry to be able to reducethe amount of pathogenic organisms and at the same time obtain optimalcharacteristics of the fermented food product e.g. acidificationprofile, flavour and colour development.

SUMMARY OF THE INVENTION

Accordingly, in interesting aspects, methods for A) manufacturing of afermented food product and B) for reducing the concentration of Listeriaspp (in particular Listeria monocytogenes in a fermented food productare provided. Said methods comprise the steps of:

-   -   (i) providing a food material,    -   (ii) mixing the food material with a starter culture,    -   (iii) mixing the food material with at least one adjunct culture        in form of a bacteriocin-producing Pediococcus species,    -   (iv) subjecting the mixture obtained in step (iii) to a        fermentation process, said fermentation process being conducted        at conditions that are sub-optimal for growth of the        bacteriocin-producing Pediococcus species in order to provide a        limited acidification and allow for a high production of        bacteriocin, and obtain the fermented food product.

In another aspect of the present invention a fermented food productobtainable by a method of the present invention is provided.

In a further aspect, the present invention provides,bacteriocin-producing Pediococcus species for use as an adjunct culturefor securing microbial safety of a fermented food product, wherein saidculture, when added to a food fermentation process, is being subjectedto conditions that are sub-optimal for growth of said species, therebyproducing bacteriocin without significantly affecting the acidificationprofile of the fermentation.

DETAILED DISCLOSURE

Prior to a discussion of the detailed aspects and embodiments of theinvention a definition of specific terms used herein is provided.

As used herein, the term “fermentation” or “food fermentation” refers tothe process of biochemical changes e.g. an acidification in animaland/or plant material (i.e. a food matrix), involving activity of livemicrobial cells under aerobic and/or anaerobic conditions to obtain afood product of desired quality.

The term “adjunct culture” is to be understood as a microbial culturethat can be added to a food matrix and produce a bacteriostatic and/orbacteriocritic product (e.g. bacteriocins and antibiotics) withoutadversely affecting the desired fermentation profile of said foodmatrix. Preferably, the adjunct culture does not adversely affect theacidification profile during manufacturing of the food product.

It follows that “conditions sub-optimal for growth” are to be understoodas growth conditions allowing the adjunct culture, when added to thefood matrix, to act as described above.

The term “starter culture” refers to a preparation containing microbialcells that is intended for inoculating a food matrix to be subjected tofermentation. The starter culture is intended for providing the desiredchange in the characteristics of the food matrix during fermentation(e.g. a desired acidification). Typically, a starter culture willproliferate during the fermentation process.

A “bioprotective agent” is to be understood as a live organism thatexerts its bioprotective effect when added to a food matrix withoutadversely affecting the food matrix.

The bioprotective effect is defined as an effect accomplished by theproduction of a bacteriostatic and/or bacteriocritic product whereby thepresence and/or activity of undesired organisms e.g. Listeriamonocytogenes is inhibited and/or diminished.

In the present context, the term “microorganism” is used in its normalmeaning. Thus, in its broadest meaning the term “microorganism” isintended to cover algae, protozoa, viruses, bacteria and fungi.Preferred microorganisms are bacteria and fungi, in particular bacteria,such as lactic acid bacteria.

The expression “lactic acid bacteria (LAB)” designates a group of Grampositive, catalase negative, non-motile, microaerophilic or anaerobicbacteria which ferment sugar with the production of acids includinglactic acid as the predominantly produced acid, acetic acid, formic acidand propionic acid. The industrially most useful lactic acid bacteriaare found among Lactococcus species, Streptococcus species, Enterococcusspecies, Lactobacillus species, Leuconostoc species, Pediococcus speciesand Bifidobacterium species.

Commonly used starter culture strains of lactic acid bacteria aregenerally divided into mesophilic organisms having optimum growthtemperatures at about 30° C. and thermophilic organisms having optimumgrowth temperatures in the range of about 40 to about 45° C. Typicalorganisms belonging to the mesophilic group include Lactococcus lactis,Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp.cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactisbiovar. diacetylactis, Lactobacillus casei subsp. casei andLactobacillus paracasei subsp. paracasei. Thermophilic lactic acidbacterial species include as examples Streptococcus thermophilus,Pediococcus acidilactici, Enterococcus faecium, Lactobacillusdelbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillusdelbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

Also the strict anaerobic bacteria belonging to the genusBifidobacterium including Bifidobacterium bifidum and Bifidobacteriumlongum are commonly used as dairy starter cultures and are generallyincluded in the group of lactic acid bacteria. Additionally, species ofPropionibacterium are used as dairy starter cultures, in particular inthe manufacture of cheese. Additionally, organisms belonging to theBrevibacterium genus are commonly used as food starter cultures.

Another group of microbial starter cultures is fungal cultures,including yeast cultures and cultures of filamentous fungi, which areparticularly used in the manufacture of certain types of cheese andbeverage. Examples of currently used cultures of fungi includePenicillium roqueforti, Penicillium candidum, Geotrichum candidum,Torula kefir, Saccharomyces kefir and Saccharomyces cerevisiae.

In the production and storage of fermented food products a persistingproblem is the potential contamination of the food material bypathogenic organisms such as Listeria spp. In order to overcome thisproblem the inventors of the present invention surprisingly found thatby applying at least one adjunct culture in form of abacteriocin-producing Pediococcus species to the food material it ispossible to reduce the amount of pathogenic organisms withoutinfluencing the fermentation profile and hence the desired sensorialquality of the food product. This effect is obtained by subjecting thebacteriocin-producing Pediococcus species to fermentation conditionsthat are sub-optimal for growth of the Pediococcus species. Typically,such conditions will be optimal for growth of the starter culture andmost surprisingly it was found that such conditions allow for a highproduction of bacteriocin by the Pediococcus species. Accordingly, thepresent invention allows the food manufacturer to select and use therecipes and processing conditions securing optimal development e.g.acidification of the starter culture and at the same time secure optimalfood safety by adding the adjunct culture of the present invention atany suitable point in time during the fermentation process.

As mentioned above, the starter culture and the adjunct culture may beadded to the food material in any order. The time lapsed between theaddition of the first of the starter culture and the adjunct culture tothe addition of the second is 0 seconds, e.g. at the most 10 seconds,such as at the most 30 seconds, e.g. at the most 1 minute, such as atthe most 5 minutes, e.g. at the most 10 minute, such as at the most 60minutes, e.g. at the most 300 minute, such as at the most 600 minutes,e.g. at the most 1 day, such as at the most 2 days, e.g. at the most 3days, such as at the most 4 days, e.g. at the most 6 days.

In a preferred embodiment of the present invention the starter cultureis added at the same time or before the adjunct culture is added to thefood material.

The invention is successful in reducing and/or inhibiting the amountand/or activity of any pathogenic organism sensitive to bacteriocinsproduced by Pediococcus spp., such as pediocin, in particular Listeriaspp., such as Listeria monocytogenes.

It has been shown that the reduction in pathogenic organisms may beprovided by mixing the food material containing the pathogenic organismswith a bacteriocin-producing Pediococcus species. Suitable Pediococcusspecies include Pediococcus pentosaceus and Pediococcus acidilactici, Inparticular, the Pediococcus acidilactici strain B-LC-20 (DSM 10313)marketed by Chr. Hansen A/S under the trademark SafePro™ is preferred.It is however contemplated that other bacteriocin-producing Pediococcusspecies may provide the same advantageous characteristics and effects asthose illustrated herein.

The effect of the bacteriocin-producing Pediococcus species is mostprobably due to the tendency of such Pediococcus species to producebacteriocins capable of killing, inactivating and/or inhibitingpathogenic organisms. Until now it has not been realized that theability of the Pediococcus species to inhibit pathogenic organisms e.g.Listeria not necessarily needs to cause a general increase inacidification when added as an adjunct culture. The optimal growthtemperature for Pediococcus species such as Pediococcus acidilactici isabout 40° C. or even higher. However, most starter cultures used forfood fermentations develop optimally, i.e. result in the desiredsensorial quality of the product, at temperatures below 30° C. Thus,previously it was not always suitable to include Pediococcus species inorder to control e.g. Listeria as a reasonable compromise betweenoptimal conditions for the starter vs. optimal conditions for thePediococcus strain could not be established. The present invention willallow the food manufacturer to freely select desired starter cultureorganisms and perform food fermentation at conditions that are optimalfor the desired development of the food product. At the same time thefood safety can be secured by adding the adjunct culture of the presentinvention without adversely influencing the fermentation profile.

Obviously, when it is realized by the skilled person that production ofbacteriocin by Pediococcus species is not linked to an increase inacidification activity it will be possible to test conditions that favorthe bacteriocin production without any significant acid production. Suchconditions, with an impact on growth of microbial organisms, are wellknown by the person skilled in the art. They include but are not limitedto water activity, atmospheric conditions, Relative Humidity (RH),nutrients such as carbon source, nitrogen source etc. and otheradditives such as minerals, vitamins etc. Thus, it is within the scopeof the present invention that conditions other than temperature, whichis used in the examples provided herein, can be used to obtain asub-optimal growth of the bacteriocin-producing Pediococcus species andthus, obtain the effect of the present invention.

In a preferred embodiment of the present invention the bacteriocinproduced by the Pediococcus species is selected from the groupconsisting of Class II bacteriocins, including bacteriocins such aspediocin, bavaracin, sakacin, curvacin, leucosin and plantaricin.

In one aspect, the present invention provides a method (A) for themanufacturing of a fermented food product.

In another aspect, the present invention provides a method (B) forreducing the concentration of Listeria spp. in a fermented food product

In the present context the term “reducing the concentration” relates toa reduction in the amount of a pathogenic organism. A reduction may beprovided by killing, inactivating or inhibiting the activity of thepathogenic organism. In an embodiment of the present invention 100% ofthe pathogenic organism are killed, inactivated or inhibited, such as atleast 90%, e.g. at least 75%, such as at least 50%, e.g. at least 40%,such as at least 30%, e.g. at least 25%, such as at least 20%, e.g. atleast 10%, such as at least 5%, e.g. at least 1%.

In certain applications, a “stabilization” of the pathogenic organismsthat may be present in the food matrix will be sufficient to render thefood safe. Thus, the adjunct culture secures that the pathogenicorganisms that are present in the food matrix do not increase innumbers.

Said methods (A and B) comprise the steps of:

-   -   (i) providing a food material,    -   (ii) mixing the food material with a starter culture,    -   (iii) mixing the food material with at least one adjunct culture        in form of a bacteriocin-producing Pediococcus species,    -   (iv) subjecting the mixture provided in step (iii) to a        fermentation process, said fermentation process being conducted        at conditions that are sub-optimal growth of the        bacteriocin-producing Pediococcus species in order to provide a        limited acidification effect abut allow for a high production of        bacteriocin, and obtain the fermented food product.

The fermented food product may be subjected to a drying processsimultaneously with the fermentation process in step (iv) and/orsubsequent to the fermentation process in step (iv) to obtain a dryfermented food product.

Several food products may be produced by the method according to thepresent invention provided that the food material is fermented. Examplesof fermented food products include, but are not limited to dairyproducts such as various cheese products, fermented meat product, suchas sausages e.g. spreadable and dried sausages and ham, fermented fishand fermented vegetables.

The fermented food product is manufactured by providing a food materialwhich is subjected to a fermentation process and optionally thefermented food product is subjected to a drying process in order toprovide a dry fermented food product.

In order to reduce the concentration of the pathogenic organisms, it isdesired that this reduction can be provided without significantlyaltering the quality of the final food product, i.e. the food producermay apply the culture to his present or preferred recipe withoutotherwise changing the recipe or processing conditions. To obtain thedesired effect a culture of bacteriocin-producing Pediococcus species isapplied to a food material as an adjunct culture, which is separatedfrom the starter culture. In the present context “adjunct culture” is aculture that is added to the food material or joined with the starterculture, but which does not form part of the starter culture, i.e. theadjunct culture is an additional culture not attempted to “produce” thefermented food product, but to supply an extra technological advantage;in this case a killing, inactivating or inhibiting effect towardspathogenic organisms. In the present context “adjunct culture” and“bacteriocin-producing Pediococcus species” are used interchangeably andadjunct culture is used to further illustrate the specificcharacteristics of the bacteriocin-producing Pediococcus species.

The manufacturing of the fermented food product is being controlled andperformed by the starter culture alone. The starter cultures isresponsible for the development of a non-limiting group of qualityparameters such as acidification, reduction in water binding and wateractivity, general appearance, colour, texture, odour, aroma, taste,flavour and other sensorial and technological parameters. Thus, minimal,or preferably no, influence on the quality parameters from the adjunctculture is provided.

In order to limit or eliminate the influence of thebacteriocin-producing Pediococcus species on the quality parameters thefermentation process is conducted at conditions sub-optimal for growthof the bacteriocin-producing Pediococcus species as describedhereinbefore.

In a specific embodiment of the present invention the optional dryingprocess is conducted at conditions that are sub-optimal for growth ofthe bacteriocin-producing Pediococcus species in order to provide alimited acidification effect and allow for a high production ofbacteriocin.

In the present context the term “limited acidification” relates to theinfluence of at least one adjunct culture on acidification. In apreferred embodiment of the present invention the limited acidificationprovides a difference in pH-value caused by the adjunct culture of 0.5pH-unit or less, such as 0.25 pH-unit or less, e.g. 0.1 pH-unit or less,such as 0.25 pH-unit or less, e.g. 0.075 pH-unit or less, such as 0.06pH-unit or less, e.g. 0.05 pH-unit or less, such as 0.04 pH-unit orless, e.g. 0.03 pH-unit or less, such as 0.02 pH-unit or less, e.g. 0.01pH-unit or less.

In a preferred embodiment of the present invention the sub-optimalgrowth conditions are provided by changing at least one of theparameters selected from the group consisting of temperature, wateractivity, RH, atmospheric composition, curing salts, added nutritions,such as the carbon source, additives, such as the chemical acidulentglucono-delta-lactone or various water binding additives and the amountsof bacteria.

In order to provide sub-optimal growth conditions in respect of theadjunct culture during the fermentation process the temperature in thefermentation process is equal to or below 30° C., such as equal to orbelow 28° C., e.g. equal to or below 26° C., such as equal to or below24° C.

In order to provide sub-optimal growth conditions during the dryingprocess in respect of the adjunct culture the temperature in the dryingprocess is equal to or below 30° C., such as equal to or below 25° C.,e.g. equal to or below 20° C., such as equal to or below 15° C., such asequal to or below 10° C., e.g. equal to or below 5° C.

When mixing the adjunct culture with the food material which eithercomprises the starter culture or which is subsequently mixed with astarter culture, the concentration of the at least one adjunct cultureincreases the inoculation level of total lactic acid bacteria at most1000 times, e.g. at most 500 times, such as at most 100 times, e.g. atmost 50 times, such as at most 10 times, e.g. at most 8 times, such asat most 5 times, e.g. at most 4 times, such as at most 3 times, e.g. atmost 2 times.

In a preferred embodiment of the present invention the at least oneadjunct culture is added in a concentration in the range of 10²-10¹⁰CFU/g product, e.g. in the range of 10²-10⁹ CFU/g product, such as inthe range of 10³-10⁹ CFU/g product, e.g. in the range of 10⁴-10⁹ CFU/gproduct, such as in the range of 10²-10⁸ CFU/g product, e.g. in therange of 10²-10⁷ CFU/g product, such as in the range of 10³-10⁷ CFU/gproduct, e.g. in the range of 10⁴-10⁷ CFU/g product, such as in therange of 10⁶-10⁷ CFU/g product, e.g. in the range of 10⁶-10⁷ CFU/gproduct such as in the range of 10⁶ CFU/g product, e.g. in the range of10³-10⁶ CFU/g product, such as in the range of 10²-10⁴ CFU/g product.

In presently preferred embodiment of the invention the adjunct cultureis added in a range of 5×10⁶-9×10⁷ CFU/g product.

In a preferred embodiment of the present invention the food materialand/or the dry fermented food product is analysed for the content ofpathogenic organisms. If the content of the organisms exceeds apredetermined acceptable level the adjunct culture may be added to thefood material in order to kill, inactivate or inhibit the pathogenicorganisms.

In the case the food material is analysed for the content of pathogenicorganisms and that it is established that the content exceeds apredetermined acceptable level the adjunct culture may be added directlyto the food material to kill, inactivate or inhibit the pathogenicorganisms.

In the case the dry fermented food product is analysed for the contentof pathogenic organisms and that it is established that the contentexceeds a predetermined acceptable level, the batches of food materialssubsequently produced are mixed with the adjunct culture to kill,inactivate or inhibit the pathogenic organisms.

Under conditions of the method of the invention thebacteriocin-producing Pediococcus species can be added to a foodfermentation without adversely affecting the fermentation profile. Thus,the species can be used as adjunct cultures for securing microbialsafety of fermented food product.

Naturally, the characteristics used to describe the method of theinvention will also apply when the strain is used as an adjunct cultureas described above.

In specific embodiments, the adjunct culture is provided in a suitablepackage. Such packages may be e.g. a pouch, a tetra-pak, a can and anyother suitable means described in the art for containing microbialspecies.

Preferably, the package or corresponding marketing material is providedwith instructions indicating the fermentation conditions that aresub-optimal for growth of the bacteriocin-producing Pediococcus species.

Further, the adjunct culture may be provided in any suitable form e.g.in a frozen or freeze dried form.

In a preferred embodiment the adjunct culture is a freeze-driedpreparation of B-LC-20 (DSM 10313) provided by Chr. Hansen A/S under thetrademark SafePro™.

The isolated strain is useful for the purposes of the inventiondescribed herein. Additionally, it is contemplated that thebacteriocin-producing Pediococcus species may be used as a bioprotectiveagent for improving safety of all food products.

Although, the invention focuses on the application of the adjunctculture during a food fermentation it is within the scope of the presentinvention that the adjunct culture can be added to a food matrix thatare not subjected to a fermentation process.

A sample of the strain has been deposited according to the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. The deposit was made on 24 Oct.1995 under the accession number DSM 10313.

The invention is further illustrated in the following non-limitingexamples and in the drawings, where

FIG. 1, A and B illustrate the pH-development during ripening ofsausages applied with or without B-LC-20 together with a Chr. HansenBactoferm™ fast fermenting starter culture. Sausages were fermented at24-20° C. for 3 days, followed by ripening at 18 to 16° C. for 11 days,and

FIG. 2. A and B illustrate pH-development during ripening of sausagesapplied with or without B-LC-20 together with a Chr. Hansen Bactoferm™traditional fermenting starter culture. Sausages were fermented at24-20° C. for 4 days, followed by ripening at 18 to 14° C. for 17 days.

EXAMPLES Example 1

Influence of B-LC-20 on pH Development in Sausage Mince.

The influence of the adjunct culture on acidification is demonstratedand a summary of the pH/time profiles encountered when applying B-LC-20to sausage mince. Table 1 shows the pH development during thefermentation period as determined every second day and table 2 shows thepH development as determined continuously from 0 to 68 hours.

TABLE 1 pH development in sausage mince determined each second day CodeDay 0 Day 2 Day 4 Day 6 Control (starter 5.79 ± 0.04 4.81 ± 0.03 4.80 ±0.04 4.79 ± 0.01 culture) Control + 5.79 ± 0.01 4.77 ± 0.01 4.77 ± 0.014.80 ± 0.04 B-LC-20

TABLE 2 pH development in sausage mince determined by continuousmeasurement Hours Code 0 10 20 30 40 50 60 68 Control (starter 5.62 5.715.58 5.16 4.97 4.86 4.79 4.77 culture) Control + 5.62 5.70 5.55 5.124.91 4.80 4.76 4.76 B-LC-20

The results show that there is no significant influence on the final pHof adding the adjunct culture in form of B-LC-20 together with thecontrol culture as compared to adding the control culture alone. B-LC-20was added in a concentration of 1.1×10⁷ CFU/g mince. Control culture wasadded in a total lactic acid bacteria concentration of 3.3×10⁶ CFU/g.

It is shown that there is a slight influence of B-LC-20 during the first20-60 hours, but the maximum difference between the two curves is 0.06pH-unit which is well within the normal batch to batch variationencountered in real sausage productions.

The control starter culture consists of a blend of lactobacilli,pediococci, micrococci and staphylococci.

Example 2

Influence of B-CL-20 on Listeria reduction

Summary of Trials

Two independent trials to assess the behavior of Listeria in sausagesalong the ripening process were performed. Three batches weremanufactured in each trial. One with the control starter culture nonactive against Listeria and two batches with the control starter culturetogether with an antilisteria starter culture added at two differentconcentrations (B-LC-20, Chr. Hansen A/S). Each batch was inoculatedwith a cocktail of five Listeria monocytogenes strains (approx. 10³CFU/g) at the time of manufacturing.

Ripening was done in an adapted versatile environment test chamber SanyoModel MLR-350H, with a fermentation period for 72 h at 14° C. with80%RH.

At the selected times three sausages were sampled from each batch fordetermining Listeria and Lactic acid Bacteria counts, pH and weightloss.

The behavior of Listeria was similar in both trials, showing importantdifferences between the control batch (A) and the batch added theantilisteria containing cultures (B and C) where Listeria diminished 2log cfu/g (trial 1) and 3 log cfu/g in trial 2. No significantdifferences in the Listeria counts between batches B and C wereobserved.

In conclusion the above result points out that the bacterial cultureB-LC-20 proved to be a suitable culture for sausages manufacturedaccording to the present formulation, showing additional Listeriareduction after the fermentation period and till the end of the ripeningas compared to the control starter culture alone.

Methodology

A Listeria challenge test in dry sausages along the ripening process wasdesigned according to the following protocol. Two independent trials(trial 1 and trial 2) were performed at Institut de Recerca I TecnologiaAgroalimentaries (IRTA), Monells, Spain. The anti-Listeria activity ofthe Lactic acid Bacteria culture B-LC-20 at two different concentrationswere tested.

Bacterial Cultures

Chr. Hansen anti.listeria culture B-LC-20 and the control starterculture was stored frozen (−20° C.) until use.

Listeria monocytogenes: Two strains were from the strain collection ofIRTA, i.e. strain CTC1011 and CTC1034; and three strains were suppliedby Chr. Hansen (P01, P05, P15). Each strain was separately grown in theIRTA standard medium “TSBYE” and stored frozen (−20° C.). Viable countswere determined before each trial in order to calculate the appropriatedilution to reach the expected inoculation in the meat mixture(approximately 10³ CFU/g).

Manufacturing

Three batches (12 Kg each) were manufactured.

Batch A Control (starter culture)+Listeria monocytogenes cocktail

Batch B Control (starter culture)+B-LC-20 low concentration

Batch C Control (starter culture)+B-LC-20 high concentration

The bacterial cultures were added separately at the time of mixing.First was added L. monocytogenes cocktail (in 20 ml saline solution),followed by the starter culture and the adjunct culture.

Ripening Conditions

Fermentation and drying of sausages were done in an adapted versatileenvironmental test chamber Sanyo Model MLR-350H.

Fermentation was carried out for 72 h at 24° C. with RH>90%.

After the fermentation period the conditions were adjusted for dryinguntil day 29 at 14° C. with 80% RH.

Analyses

During fermentation and drying, pH and weight loss were measured in 3marked sausages per batch, daily during the first week and with aninterval of 3-4 days during the last 3 weeks.

Microbial Analyses

Three different sausages from each batch (A, B, C) were nalaysed at eachsampling time (days: zero (after 4 hours), 2, 7, 14 and 29). Each sampleconsisted of 25 grams of a previous homogenized sausage.

The determination of Listeria was done at each sampling time, except atday 0, by Most Probably Number technique (MPN) in Fraser Broth Base(Oxoid)+Half Fraser selective supplement (Oxoid) (48 hours, 37° C.)followed by confirmation of the positive tubes in Palcam ListeriaSelective Agar Base (Merck)+Selective Supplement att. Van Netten et al.(Merck) (48 hours, 37° C.).

Listeria counts at time zero were done by spreading the appropriatedilutions in Palcam supplemented agar plates and incubating at 37° C.for 72 hours. Lactic acid bacteria counts were performed at eachsampling time in MRS agar (72 hours at 30° C. under anaerobicconditions).

TABLE 1 Listeria spp. And Lactic Acid Bacteria counts along the ripeningof dry sausages in trial 1. Lactic Acid Batch Time (days) Listeria spp.Bacteria A 0 3.31 ± 0.02 6.47 ± 0.11 A 2 2.69 ± 0.33 8.85 ± 0.05 A 73.15 ± 0.19 8.93 ± 0.07 A 14 2.45 ± 0.18 8.73 ± 0.06 A 29 2.34 ± 0.358.60 ± 0.01 B 0 3.25 ± 0.04 7.50 ± 0.06 B 2 2.24 ± 0.12 8.73 ± 0.08 B 71.77 ± 0.35 8.78 ± 0.17 B 14 1.18 ± 0.39 8.61 ± 0.03 B 29 0.89 ± 0.698.51 ± 0.02 C 0 3.22 ± 0.10 7.77 ± 0.05 C 2 2.05 ± 0.41 8.68 ± 0.07 C 71.48 ± 0.26 8.75 ± 0.20 C 14 1.43 ± 0.51 8.52 ± 0.04 C 29 0.54 ± 0.168.41 ± 0.09 Values are the average of triplicate samples expressed aslog cfu/g standard deviation.

Batches were inoculated as follows:

Batch A (control starter culture, 3.0×10⁶ CFU/g), Batch B (controlstarter culture+B-LC-20 low concentration (2.9×10⁷ CFU/g mince)), BatchC (control starter culture+B-LC-20 high concentration (5.6×10⁷ CFU/gmince)).

All the batches were inoculated with a cocktail of 5 different Listeriamonocytogenes strains (CTC1011, CTC1034, P01, P05, P15).

TABLE 2 pH and weight loss along the ripening of dry sausages intrial 1. Batch Time (days) pH Weight loss % A 0 5.82 ± 0.01 NA A 1 5.57± 0.04  1.54 ± 0.73 A 2 4.90 ± 0.09  2.96 ± 1.49 A 3 4.67 ± 0.04  5.79 ±1.13 A 4 4.60 ± 0.01 10.36 ± 0.79 A 7 4.69 ± 0.01 17.04 ± 0.60 A 11 4.75± 0.01 21.66 ± 0.54 A 14 4.71 ± 0.01 24.31 ± 0.34 A 18 4.86 ± 0.02 27.04± 0.38 A 24 4.75 ± 0.01 30.23 ± 0.33 A 29 4.99 ± 0.13 31.89 ± 0.36 B 05.79 ± 0.03 NA B 1 5.50 ± 0.05  1.22 ± 0.64 B 2 4.92 ± 0.01  2.46 ± 0.76B 3 4.61 ± 0.03  4.24 ± 0.74 B 4 4.49 ± 0.01  8.71 ± 1.15 B 7 4.55 ±0.02 15.92 ± 0.71 B 11 4.58 ± 0.01 21.71 ± 0.33 B 14 4.49 ± 0.17 25.02 ±0.65 B 18 4.69 ± 0.01 28.36 ± 0.39 B 24 4.63 ± 0.01 31.27 ± 0.58 B 294.75 ± 0.01 32.89 ± 0.61 C 0 5.83 ± 0.01 NA C 1 5.37 ± 0.01  1.32 ± 0.49C 2 4.89 ± 0.03  2.67 ± 0.53 C 3 4.59 ± 0.02 5.104.89 ± 0.03   C 4 4.49± 0.01  9.66. ± 0.33 C 7 4.55 ± 0.01 16.69. ± 0.26  C 11 4.61 ± 0.0122.57 ± 0.30 C 14 4.57 ± 0.02 25.57 ± 0.28 C 18 4.69 ± 0.01 28.60 ± 0.07C 24 4.62 ± 0.01 31.56 ± 0.25 C 29 4.74 ± 0.03 33.15 ± 0.26 NA = nonapplicable Values are the average of triplicate samples ± standarddeviation.

TABLE 3 Listeria spp. and Lactic Acid Bacteria counts along the ripeningof dry sausages in trial 2. Lactic Acid Batch Time (days) Listeria spp.Bacteria A 0 3.40 ± 0.01 6.41 ± 0.15 A 2 2.56 ± 0.54 8.83 ± 0.04 A 72.68 ± 0.36 8.75 ± 0.04 A 14 1.86 ± 0.19 8.76 ± 0.03 A 29 1.56 ± 0.358.39 ± 0.14 B 0 3.39 ± 0.03 7.25 ± 0.07 B 2 2.04 ± 0.12 8.56 ± 0.10 B 71.13 ± 0.26 8.58 ± 0.11 B 14 1.16 ± 0.20 8.35 ± 0.08 B 29 0.33 ± 0.178.33 ± 0.08 C 0 3.67 ± 0.05 7.79 ± 0.02 C 2 1.87 ± 0.19 8.50 ± 0.08 C 71.56 ± 0.35 8.47 ± 0.07 C 14 0.84 ± 0.20 8.52 ± 0.12 C 29 0.23 ± 0.238.34 ± 0.04 Values are the average of triplicate samples as log cfu/gstandard deviation.

Batches were inoculated as follows:

Batch A (control starter culture, 2.6×10⁶ CFU/g), Batch B (controlstarter culture+B-LC-20 low concentration (1.5×10⁷ CFU/g), Batch C(control starter culture+B-LC-20 high concentration (5.9×10⁷ CFU/g).

All the batches were inoculated with a cocktail of 5 different Listeriamonocytogenes strains (CTC1011, CTC1034, P01, P05, P15).

TABLE 4 pH and weight loss along the ripening of dry sausages in trial2. Batch Time (days) PH Weight loss % A 0 6.10 ± 0.07 NA A 1 5.83 ± 0.02 3.09 ± 1.27 A 2 5.04 ± 0.03  4.73 ± 1.13 A 3 4.86 ± 0.03  7.30 ± 1.39 A4 4.90 ± 0.01 10.03 ± 0.89 A 7 4.91 ± 0.01 14.81 ± 0.76 A 11 4.95 ± 0.0119.19 ± 0.53 A 14 4.94 ± 0.01 21.14 ± 0.48 A 18 4.98 ± 0.01 24.08 ± 0.66A 24 5.04 ± 0.01 27.41 ± 0.44 A 29 5.04 ± 0.01 29.92 ± 0.43 B 0 5.97 ±0.02 NA B 1 5.52 ± 0.07  3.79 ± 1.48 B 2 4.98 ± 0.02  5.17 ± 1.03 B 34.72 ± 0.03  7.90 ± 1.70 B 4 4.72 ± 0.01 11.31 ± 1.06 B 7 4.71 ± 0.0316.53 ± 0.95 B 11 4.74 ± 0.01 21.84 ± 0.38 B 14 4.75 ± 0.01 23.96 ± 0.39B 18 4.80 ± 0.02 27.24 ± 0.40 B 24 4.84 ± 0.02 30.90 ± 0.20 B 29 4.89 ±0.02 33.26 ± 0.13 C 0 5.99 ± 0.01 NA C 1 5.49 ± 0.04  2.77 ± 0.59 C 24.93 ± 0.01  4.16 ± 1.16 C 3 4.68 ± 0.01  6.33 ± 0.75 C 4 4.70 ± 0.019.985 ± 0.48 C 7 4.68 ± 0.02 15.84 ± 0.60 C 11 4.70 ± 0.01 21.29 ± 0.61C 14 4.74 ± 0.01 23.52 ± 0.51 C 18 4.79 ± 0.02 26.65 ± 0.62 C 24 4.84 ±0.02 30.37 ± 0.58 C 29 4.83 ± 0.02 32.69 ± 0.67 NA = non applicableValues are the average of triplicate samples ± standard deviation.

Discussion

Two independent trials to assess the behavior of Listeria in sausagesalong the ripening process were performed. Three batches weremanufactured in each trial. One control with starter culture non activeagainst listeria and two batches with the same starter culture plus anantilisteria adjunct culture added at two different concentrations(B-LC-20) in each batch. Each batch was inoculated with a cocktail offive Listeria monocytogenes strains (approx. 1 03 cfu/g) at the time ofmanufacturing.

In both trials and after 2 days of fermentation Listeria diminished. Thecounts in batch B and C were lower than in batch A. These differencesbetween the control batch A and the batches inoculated with theantilisterial cultures increased until the end of ripening. In trial 1,by the end of the ripening, Listeria diminished 1 log cfu/g in batch Awhile in batch B and C Listeria decreased more than 2 logs cfu/g. Intrial 2 by the end of ripening Listeria diminished 1.8 (log cfu/g) inbatch A, and more than 3 logs in batch B and C.

Lactic acid bacteria counts reached the maximum after 2 days offermentation with similar values at the end of the process in each batch(around 108 cfu/g) in both trials. The curve of pH was similar for thedifferent batches in both trials. The minimum pH was recorded after 4days in trial 1 and after 3 days in trial 2 despite the pH at time zerowas higher in trial 2. The pH-drop in the control batches were in bothtrials similar to the pH-drop in the batch with added adjunct culture.ΔpH was between 1.22-1.24 in batch A after 3-4 days of fermentation, andbetween 1.26-1.34 in batches B and C. The small differences wereprobably caused by the extra glucose added with the adjunct culturepouch. Weight loss showed similar profile in both trials with nodifferences between the lots.

CONCLUSION

The culture B-LC-20 proved to be a suitable protective adjunct culturefor fermented sausages manufactured according to the presentformulation, showing additional Listeria reduction after thefermentation period and till the end of ripening, compared to a controlstarter culture alone.

In general, addition of extra inoculum of lactic acid bacteria reducesthe time to on-set of fermentation (the lag phase) and thereby speed upthe overall acidification rate. When increasing the inoculum by 10 times(from 5·10⁵ to 5·10⁶ CFU/g) acidification lag phase for a typical NorthEuropean type fermented sausage was halfed and the time to reach pH of5.3 and 4.9 reduced by 25 and 30%, respectively.

Addition of B-LC-20 to the sausage recipe in Example 2, trial 1 resultedin increased inoculation level of total lactic acid bacteria of approx.15 times, from 3.10⁶ to between 3·10⁷-6·10⁷. It was expected that thelag phase would have been reduced considerably and the time to reach pH4.9 reduced by at least 30%. This expected reduction did not take place,pH reached 4.9 after 2 days for all three batches, i.e. addition of anadjunct culture such as B-LC-20 to the existing recipe did not speed upacidification time as expected. In example 1, table 1 and 2acidification speed did not increase significantly, either.

Therefore, the inventors of the present invention surprisingly foundthat the use of an adjunct culture such as B-LC-20 provides a uniqueanti-listerial reduction for fermented sausages since it was found thatPediococcus acidilactici is a strong producer of pediocin (whichdestroys Listeria monocytogenes) at European fermentation temperatures(<26 ° C.) while not being a strong acidifier at this temperature.

The reduction of Listeria is primarily caused by pediocin produced by anadjunct culture such as B-LC-20 in the food material during thefermentation and drying process. The effect of pediocin is a well knownphenomenon in the literature. However, the uniqueness of the adjunctculture (B-LC-20) and the method disclosed herein is that the foodmanufacturer can use the adjunct culture together with the normalacidification culture since it does not alter the overall acidificationprofile and the quality of the product significantly. As mentionedabove, the acidification profile is of utmost importance for the sensoryquality. Thus, the manufacturer does not need to change his presentrecipe or processing conditions, but will get the advantage of reductionin Listeria numbers.

Example 3

Influence of the Adjunct Culture on the Acidification Profile ofDifferent fermented Sausages

The influence of the adjunct culture B-LC-20 on the acidificationprofile of four types of sausages fermented with different startercultures are demonstrated in FIGS. 1 and 2. In all cases, the additionof the adjunct culture in the sausage mince together with the starterculture did not influence the acidification profile of the sausagessignificantly. In addition, internal sensory evaluations showed that thesensory quality of the sausages was unchanged by addition of B-LC-20.

Organisms Inoculation, total LAB F-1 Pediococcus pentosaceus 5 × 10⁶CFU/g mince Staphylococcus xylosus F-SC-111 Lactobacillus sakei 1 × 10⁷CFU/g mince Staphylococcus carnosus T-SPX Pediococcus pentosaceus 5.5 ×10⁶ CFU/g mince   Staphylococcus xylosus T-SC-150 Lactobacillus sakei 1× 10⁷ CFU/g mince Staphylococcus carnosus

1-22. (canceled)
 23. A method for suppressing the growth of microbialpathogens in a fermented food product without altering its acidificationprofile, comprising: (i) providing a food material; (ii) mixing the foodmaterial with a starter culture wherein the started culture provides thedesired change in the characteristics of the food material duringfermentation; (iii) mixing the food material with at least one adjunctculture comprising a bacteriocin-producing Pediococcus acidilactici;(iv) subjecting the food material containing the starter culture andadjunct culture to a fermentation process at a temperature not more than30° C., wherein the fermentation process (A) results in acidification ofthe food material, of which the acidification caused by the adjunctculture is 0.5 pH-unit or less; and (B) allows for a production ofbacteriocin in an amount sufficient to reduce counts of Listeria by atleast 2 log cfu/g of fermented food product.
 24. The method of claim 23,wherein the fermentation process is conducted at a temperature not morethan 25° C.
 25. The method of claim 23, further comprising a dryingprocess simultaneously with the fermentation process in step (iv) and/orsubsequent to the fermentation process in step (iv) to obtain a dryfermented food product.
 26. The method according to claim 23, whereinthe acidification caused by the adjunct culture is 0.25 pH-unit or less.27. The method of claim 26, wherein the fermentation process isconducted at a temperature not more than 28° C.
 28. The method of claim23, wherein the starter culture comprises lactic acid bacteria and theaddition of the adjunct culture increases the number of total lacticacid bacteria at least 1000 times compared to the number without theadjunct culture.
 29. The method of claim 23, wherein the adjunct cultureis added in a concentration in the range of 10²-10¹⁰ CFU/g foodmaterial.
 30. The method of claim 23, wherein the adjunct culture isadded in a concentration of 2×10⁷ CFU/g food material.
 31. The method ofclaim 23, wherein the fermented food product is a fermented dairyproduct.
 32. The method of claim 23, wherein the bacteriocin-producingPediococcus acidilactici strain is Pediococcus acidilactici strain DSM10313.
 33. The method of claim 23, wherein the starter culture comprisesa lactic acid bacteria and at least one bacteria selected from thegenera Micrococcus and Staphylococcus.
 34. The method of claim 23,wherein the starter culture comprises at least one organism selectedfrom the group consisting of Lactococcus lactis, Lactococcus lactissubsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcuspentosaceus, Lactococcus lactis subs p. lactis biovar. diacetylactis,Lactobacillus casei subs p. casei, Lactobacillus paracasei subsp.paracasei, Bifidobacterium bifidum, Bifidobacterium longum,Propionibacterium spp. Brevibacterium spp. Penicillium roqueforti,Penicillium candidum, Geotrichum candidum, Torula kefir, Saccharomyceskefir, and Saccharomyces cerevisiae.